Stirring and mixing liquids is a necessary part of many industrial, chemical and pharmaceutical technological processes. The majority of these industrial processes are carried out on macroscopic levels. It has only been in the recent years that mixing of small quantities of liquids has become technologically relevant in the context of microfluidics since mixing is often crucial to the effective functioning of devices manipulating with small quantities of liquids. (Nguyen, N. & Werely, S. 2002 Fundamentals and applications of microfluidics. Boston, Mass.). Microfluidic devices are useful in various biological and chemical applications, including such diverse fields as biochemical analysis, drug screening, genetic analysis, medical diagnostics, chemical synthesis, and environmental monitoring. One exemplary important application of microfluidics is in biochemical sensing techniques such as immunoassays and hybridization analyses, which require rapid, homogeneous mixing of macromolecular solutions, such as DNA or proteins. Achieving effective stirring and mixing in macroscopic volumes of fluids is a relatively straightforward task. Various conventional mechanically or magnetically driven stirring elements may be employed. Alternatively, special geometries may be employed in flow channels to promote mixing without the use of moving elements.
Stirring and mixing in small volumes is, however, difficult. Applying conventional mixing strategies to microfluidic volumes is generally ineffective. Various designs of micromixers have been proposed in recent years. There are several publications that comprehensively review mixing methods and devices developed for microfluidic applications (see for example Christopher J. Campbell and Bartosz A. Grzybowski. Microfluidic mixers: from microfabricated to self-assembling devices. Phil. Trans. R. Soc. Lond. A (2004) 362, 1069-1086; and Julio M. Ottino and Stephen Wiggins. Introduction: mixing in microfluidics. Phil. Trans. R. Soc. Lond. A (2004) 362, 923-935).
Mixing methods are usually classified as either passive or active. Passive mixers have no moving parts and achieve mixing by virtue of their topology alone, while active mixers either do have moving parts or they use externally applied magnetic, electromagnetic or acoustic field. For example, U.S. Pat. Nos. 6,877,892; 6,890,093 and 6,935,772 issued to Karp et al. disclose devices for mixing multiple fluid streams passively using structures such as channel overlaps, slits, converging/diverging regions, turns, and/or apertures. The devices include microfluidic channels that are formed in various layers of a three-dimensional structure. U.S. Patent Application No. 20060280029 filed by Garstecki et al. discloses a microfluidic mixer that includes a channel having an inlet that separates into at least two branches, the branches then recombining into a single outlet.
Although performance of these devices is in many cases satisfactory, their fabrication is usually a tedious, multi-step process. The lack of moving parts makes passive mixers free of additional friction and wear effects, but their intricate channel topologies are often hard to fabricate, and they are generally not switchable: once incorporated into a fluidic system, they perform their function whenever fluids pass through them.
In contrast, active mixers can be controlled externally, which makes them suitable as components for reconfigurable microfluidic systems: that is, systems that can perform several different functions given different states of external controls. Active micromixers are known to be of two types: with and without moving parts. The moving parts can be microscopic stirrer bars, piezoelectric membranes or oscillating gas bubbles. The mixing can be achieved also without moving parts by action of electrical or acoustic fields on the liquid. U.S. Pat. No. 7,081,189 issued to Squires et al. discloses one example of a microfluidic mixer driven by induced-charge electro-osmosis applied to electrolyte fluids. Liu et al. developed an approach to micromixing based on acoustic microstreaming around an array of small air bubbles resting at the bottom of the mixing chamber (Liu, R., Lenigk, R., Druyor-Sanchez, R. L., Yang, J. & Grodzinski, P. 2003 Hybridization enhancement using cavitation microstreaming. Analyt. Chem. 75, 1911-1917). When the bubbles were made to vibrate by a sound field, they created steady circular flows around them. U.S. Pat. No. 6,244,738 issued to Yasuda et al. discloses ultrasonic vibrators arranged in the stirring tube where plural sample solutions to be mixed are stirred and mixed by an acoustic streaming induced by ultrasonic vibration.
One of the areas where microstirring is important is in the bead-based immunoassays, such as the latex agglutination test (LAT) used for identification and quantification of analytes, biomolecules and other substances of biological importance. LAT is widely used in point-of-care tests for diagnostic purposes, as well as in drug discovery/proteomics research, and in food-industry quality controls due to its simplicity, low cost and speed. There are several drawbacks of the particle agglutination methods such as long time of analysis dictating therefore the need for mechanical rotational motion of glass slides to accelerate the agglutination process; and a limited analytical sensitivity of the assay because of formation of nonspecifically bound aggregates. Effective microstirring may enhance bead-based assay by first accelerating immunochemical reaction and then by destroying nonspecifically bound aggregates and improving signal-to-noise ratio in quantitative assessment of the amount of immunochemically bound aggregates.
It is known to employ acoustic energy and specifically the phenomenon of a standing wave to manipulate particles suspended in a fluid, for example, to separate different types of particles from a liquid or from each other. The use of a nodal pattern of a standing wave associated with a single resonance frequency for particle capture and manipulation is described in detail for various patents, for example as listed below (these patents are incorporated herein in their entirety by reference):
4,055,4914,280,8234,398,9254,523,6324,523,6824,673,5124,759,7754,877,5164,879,0115,006,2665,527,4605,613,4565,626,7675,688,406as well as in the US Patent Application No. 2006037915 and international application No. PCT/AT89/00098.
The forces responsible for redistributing particles in the liquid in accordance with the nodal pattern of an ultrasonic standing wave depend on the relative density and acoustic impedance of the particles with respect to the fluid in which they are suspended, the dimensions of the particles and the frequency of the standing ultrasonic wave. Ultrasound radiation force drives the particles to the local particle potential energy minima within the pressure nodal planes, to give concentration regions that appear as clumps striated at half-wavelength separation (W. L. Nyborg, Mechanisms for nonthermal effects of sound, J. Acoust. Soc. Am., 1968, 44, 1302-1309; Wiklund M, Hertz H M. Ultrasonic enhancement of bead-based bioaffinity assays. Lab Chip. Oct. 6, 2006 (10):1279-92, incorporated herein in its entirety by reference).
Although the use of a nodal pattern of a single resonance frequency standing wave is well described in the prior art for capturing and manipulating particles, there is no mentioning of using swept-frequency mode of generation of standing waves of multiple frequencies for liquid stirring and mixing.
Thus, there is a need for a device capable of thoroughly and rapidly mixing small volumes of fluids in a microfluidic environment.